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Bone is a dynamic organ that undergoes continuous remodeling. Over-activated osteoclasts activity can lead to osteolytic bone conditions, resulting in several bone diseases such as osteoporosis. Thus, the inhibition of osteoclastic activity and the resultant bone resorption have a profound effect on regulating abnormal remodeling processes and is therapeutically important for osteoporosis prevention. Hence, targeting NF-κB and MAPK/activator protein 1 (AP-1) signaling pathways to inhibit osteoclastogenesis has been considered a promising strategy for osteoporosis treatment. To date, most FDA-approved medications against osteoporosis are associated with severe side effects such as renal system impairment. Food-derived natural products are gaining the momentum in the development of alternative treatment options against osteoporosis. Ovotransferrin is an iron-binding glycoprotein, which constitutes about 12% of the total egg white protein. The aim of this work was to characterize the cellular and molecular functions of egg white ovotransferrin on osteoclasts differentiation and function.
The murine monocyte cell line RAW 264.7 (ATCC TIB-71) were cultured in DMEM supplemented with 10% FBS and 1% pen-strep at 37 °C with 5% CO2 atmosphere. Osteoclast cells were induced from murine monocyte cell line RAW 264.7. RAW 264.7 cells. Osteoclasts were successfully induced by incubating with RANKL for 4 days. RAW 264.7 cells were treated with different concentrations of ovotransferrin (1 to 1000 μg/mL) for 4 days to examine the effects of ovotransferrin on osteoclastogenesis. The cells were observed with a light microscope under a 10× lens. For the Resorption Assay, RAW 264.7 cells were incubated in a calcium phosphate (CaP)-coated 48-well plate with 100 ng/mL RANKL to generate multinucleated osteoclasts, and treated with 1–1000 μg/mL ovotransferrin for 6 days. The generated multinucleated osteoclasts were identified with microscopy every day.
The total number of TRAP-positive (TRAP+) osteoclasts confirmed the inhibition effect of ovotransferrin on RANKL-induced osteoclast differentiation, although only high doses of ovotransferrin (100 and 1000 μg/mL) produced significant effects. At high concentrations (100 and 1000 μg/mL), ovotransferrin significantly inhibited RANKL-induced phosphorylation of MAPKs. These data indicate that ovotransferrin inhibits osteoclast formation and differentiation by attenuating the activation of NF-κB and MAPK pathways.
In this study, we established that egg white ovotransferrin could suppress RANKL-mediated osteoclastogenesis and resorption activity via suppression of NF-κB and MAPK activation during the process of osteoclast differentiation along with the induction of cell apoptosis in mature osteoclasts. We found that the egg white protein ovotransferrin inhibits osteoclasts differentiation and survival, suggesting its potential for use as a functional food ingredient in bone health management.